CD64, also known as the type I Fc-gamma receptor, functions as a high affinity receptor for IgG. It is involved in the activation of phagocytosis, endocytosis of IgG-opsonized particles, and activation of the complement system. Our goal is to determine the structure of CD64 and its complex with immunoglobulins. To express and purify the recombinant CD64 protein, we have constructed both bacteria and a CHO cell based expression system. The yield of the CHO cell expression remained at a level of 50 ug/liter of cell culture after gene amplification. The bacteria system, on the other hand, yielded the protein in an inactive inclusion body form. Subsequent refolding experiments resulted in less than a mg of the protein for crystallization. Currently, we are attempting to express the recombinant CD64 using a baculovirus expression system. B cells initiate their signaling through the formation of antigen cross-linked B cell receptor (BCR) which consists of an immunoglobulin (Ig) molecule and Ig alpha (Iga) and Ig beta (Igb). In efforts to elucidate the interaction between Iga and Igb and between the heterodimer and the BCR, we have expressed the extracellular domains of Iga and Igb in E. coli as inclusion bodies. Each protein was encoded in a pET-30 vector and included a C-terminal 6-His tag for purification, and high yields of both Iga and Igb inclusion bodies were obtained (15-50 mg/L of culture). Attempts were made to refold Iga and Igb either individually or in concert. Refolding of Iga alone yielded approximately 2 mg of pure protein from 50 mg of inclusion bodies following purification by Ni-NTA and gel filtration chromatography. The purified protein was present as a mixture of monomeric and homodimeric Iga as determined by mass spectrometry and SDS-PAGE analysis; the Iga-Iga dimer was most likely noncovalent. Purified Iga was used for antibody production and was also subjected to crystallization trials. However, the sample appeared to be unstable, precipitating above 4?C, and no hits were obtained with crystallization screens. Refolding of Igb alone failed to yield significant quantities of protein due to degradation. Initial attempts to co-refold Iga and Igb together by standard dilution refolding methods were also plagued by degradation of Igb. However, by sequestering Iga and Igb inclusion bodies together in a smaller volume during refolding, we purified by Ni-NTA affinity chromatography and anion exchange a mixture of monomeric and dimeric Iga, and the Iga-Igb heterodimer. We are also expressing the murine Iga-Igb using a bacteria expression/refolding system.